biochemical pharmacology

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3 The design of redox active thiol peroxidase mimics: Dihydrolipoic acid4 recognition correlates with cytotoxicity and prooxidant action

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7 B. Zadehvakili a, S.M. McNeill b, J.P. Fawcett a, G.I. Giles b,⇑8 a School of Pharmacy, University of Otago, Dunedin, New Zealand9 bDepartment of Pharmacology and Toxicology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand

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1 3a r t i c l e i n f o

14 Article history:15 Received 13 November 201516 Accepted 14 January 201617 Available online xxxx

18 Keywords:19 Drug design20 Glutathione peroxidase21 Dihydrolipoic acid22 Organoselenium23 Organotellurium24 Redox25

2 6a b s t r a c t

27Redox active molecules containing organoselenium or organotellurium groups catalyse the oxidation of28cellular thiols by hydrogen peroxide and are currently being developed as therapeutic agents. Potentially29these synthetic thiol peroxidase (TPx) mimics can protect cells from oxidative stress by catalysing the30reduction of reactive oxygen species by the cellular thiol glutathione, an activity which mimics the func-31tion of the antioxidant enzyme glutathione peroxidase. Alternatively they can act as prooxidants by cat-32alysing the oxidation of essential thiol species within the cell. However the structure–activity33relationships which determine the choice of thiol substrate, and hence the overall antioxidant or proox-34idant outcome of drug administration, remain unknown. We report the first study that relates the phar-35macological properties of TPx mimics with their solubility and catalytic activity using different thiol36substrates. We used a series of structurally related compounds PhMCnH2n+1 (M = Se, Te; n = 4–7) and37investigated their ability to catalyse the oxidation of the cellular thiols glutathione and dihydrolipoic acid38by hydrogen peroxide. The resulting rate constants (kobs) were then related to compound cytotoxicity and39antioxidant versus prooxidant action in A549 cancer cells. The results show that the dihydrolipoic acid40kobs values correlate with both cytotoxicity and prooxidant function. This enabled us to define a relation-41ship, IC50 = 10 + 280e�5(DHLA kobs), which allows the prediction of TPx mimics cytotoxicity. In contrast,42hydrophobicity and glutathione kobs were unrelated to the compounds’ redox pharmacology.43� 2016 Elsevier Inc. All rights reserved.44

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48 1. Introduction

49 Oxidative stress is a common feature of many pathologies.50 Abnormal levels of reactive oxygen species (ROS) have been iden-51 tified in over 150 diseases including Alzheimer’s Disease [70],52 myocardial infarction [35] and cancer [36,50]. Due to the wide-53 spread involvement of the cellular redox state in multiple diseases,54 drug development programmes are focused on developing ‘‘redox55 modifying” molecules which either scavenge or generate ROS as56 a means of treating redox linked morbidities. However the field

57is in its infancy and the cellular redox environment is poorly58understood [34]. As a result only a few redox active drugs have59entered the clinic, although others are progressing through clinical60trials [1,59,68]. To develop these new therapeutic entities there is a61need to improve our understanding of the mechanisms by which62redox modifying drugs interact with the cellular redox state and63hence modify disease outcomes.64Of longstanding interest as potential redox drugs are molecules65which incorporate an organoselenium or organotellurium group66and mimic the selenocysteine based enzyme glutathione peroxi-67dase (GPx) [44,45,60]. Endogenous GPx acts as a protective antiox-68idant enzyme which neutralises the toxic effects of hydrogen69peroxide (H2O2) and lipid peroxides by catalysing their reduction.70In its catalytic cycle GPx uses the abundant cellular thiol glu-71tathione (GSH) as a co-substrate, oxidising GSH to the disulphide72(GSSG), while concomitantly reducing a peroxide species to water73(for H2O2) or a lipid alcohol (for lipid peroxides [45,60]). By scav-74enging H2O2, GPx protects other essential species from oxidation75and so preserves cell function [2].

http://dx.doi.org/10.1016/j.bcp.2016.01.0120006-2952/� 2016 Elsevier Inc. All rights reserved.

Abbreviations: DHLA, dihydrolipoic acid; DMEM, Dulbecco’s Modified Eagle’sMedium; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine serum; GPx,glutathione peroxidase; GSH, glutathione; GSSG, glutathione disulphide; HPLC,high performance liquid chromatography; KPi, potassium phosphate; MTT,methylthiazolyldiphenyl tetrazolium bromide; PBS, phosphate buffered saline;ROS, reactive oxygen species; SAR, structure–activity relationship; TPx, thiolperoxidase.⇑ Corresponding author at: Department of Pharmacology and Toxicology, Univer-

sity of Otago, P.O. Box 913, Dunedin, New Zealand.E-mail address: [email protected] (G.I. Giles).

Biochemical Pharmacology xxx (2016) xxx–xxx

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Please cite this article in press as: B. Zadehvakili et al., The design of redox active thiol peroxidase mimics: Dihydrolipoic acid recognition correlates withcytotoxicity and prooxidant action, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.01.012

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76 The first organoselenium compound to be classified as a GPx77 mimic was ebselen [45], which has undergone clinical trial for78 stroke [69]. Since ebselen’s introduction, a range of analogues [4]79 as well as structurally diverse organoselenium [9,40,57,67] and80 organotellurium based compounds [33] have been evaluated in81 various disease models. Nevertheless, the pharmacological mecha-82 nisms involved in the action of this family of compounds have pro-83 ven difficult to define. While in vitro studies have shown that the84 molecules can mimic GPx by catalysing the reaction of GSH with85 peroxides [39], they lack the substrate specificity of the native86 enzyme [20,41] and can accept an array of thiols as substrates87 [30]. These drugs are therefore more accurately referred to as thiol88 peroxidase (TPx) mimics rather than GPx mimics.89 The ability to oxidise a range of thiols has important mechanis-90 tic implications, since it can involve the oxidation of cysteine con-91 taining proteins resulting in changes to their structure and92 function. Other targets within the cellular thiol proteome include93 transcription factors [8], components of signal transduction path-94 ways [13], and enzymes regulating the production of metabolites95 and inflammatory mediators [66]. Therefore TPx mimics can act96 as either antioxidants or prooxidants depending on their thiol97 specificity [38]. Currently the structure–activity relationships98 (SARs) which determine thiol recognition are completely99 unknown, which greatly limits the development of these mole-

100 cules as therapeutics.101 To address this limitation we have recently developed an elec-102 trode based assay to measure TPx activity [26]. The assay uses an103 H2O2 selective electrode to monitor the rate of H2O2 reduction,104 rather than the rate of thiol oxidation, and can thereby determine105 rate constants (kobs) for any given TPx mimic with a range of thiol106 substrates. The present study investigates the application of this107 assay to define SARs for TPx mimic cytotoxicity and antioxidant108 versus prooxidant action. We selected groups of compounds con-109 taining a Se or Te atom asymmetrically substituted with an aro-110 matic phenyl ring and a normal alkyl chain (Fig. 1). This111 pharmacophore is known to possess TPx activity [14,15,26,58]112 and stability to decomposition via the telluroxide elimination reac-113 tion [54]. The resulting pool of compounds was sub-divided into S114 (Se) and T (Te) subsets, depending on the catalytic metal centre.115 To assess TPx activity (Fig. 2) towards pharmacologically rele-116 vant thiol species, we determined rate constants with GSH (GSH117 kobs) and dihydrolipoic acid (DHLA kobs). GSH is an endogenous118 cytosolic antioxidant [11] while DHLA is a naturally occurring119 dithiol found in the mitochondria, where it acts as a cofactor for120 enzymes involved in energy metabolism [27]. DHLA is also present121 in the cytosol and plasma membrane, where it functions as an122 antioxidant [3,42]. While DHLA is present at much lower cellular123 concentrations than GSH, it has previously been characterised as124 a superior substrate for ebselen [28], indicating it may be a rele-125 vant substrate for TPx mimics. This was followed by the analysis126 of compound cytotoxicity, and the ability of the TPx mimics to127 act as either antioxidants or prooxidants versus hydrogen peroxide128 induced oxidative stress. For these studies we utilised the A549

129lung carcinoma cell line as a commonly utilised model system130for the investigation of drug cytotoxicity [12] and metabolism131[21]. We report the effect that increasing the alkyl chain length,132as well as substituting Se for Te, had on TPx catalytic activity, cyto-133toxicity and antioxidant versus prooxidant action. This is the first134SAR study to investigate these properties in relation to thiol135substrate.

1362. Materials and methods

1372.1. Materials

138The A549 lung carcinoma cell line was obtained from the ATCC139(Manassas, VA, USA). DHLA, dimethylsulphoxide (DMSO), EDTA,140GSH, H2O2 and methylthiazolyldiphenyl tetrazolium (MTT) were141purchased from Sigma–Aldrich (Auckland, New Zealand). Antibi-142otic–antimycotic solution, Dulbecco’s Modified Eagle’s Medium143(DMEM), foetal bovine serum (FBS), phosphate buffered saline144(PBS) and trypsin were purchased from Life Technologies (Auck-145land, New Zealand). Electrochemical measurements were per-146formed in a sealed four port chamber using a TBR4100 Free147Radical Analyser system (WPI, Sarasota, USA) with a 2 mm diame-148ter ISO-HPO-2 H2O2 selective sensor electrode. Chemical character-149isation of TPx mimics was performed using a Prospero-V 500 MHz150NMR and a Jenway 6715 UV–Vis spectrometer. HPLC was per-151formed using a Shimadzu LC-10AT HPLC system equipped with a152SIL-10AD autoinjector, SPD-M10A diode array detector, and Gem-153ini C18 column (5 lm, 100 Å, 100 � 4.6 mm). Fluorescence live cell154imaging was performed using an inverted fluorescence microscope155(Nikon Ti-U Eclipse, Coherent Scientific, Hilton, SA, Australia).

1562.2. Synthesis of TPx mimics

157TPx mimics were prepared according to an established proce-158dure [17]. Compound structure was confirmed by comparing 1H159NMR and 13C NMR spectra with published data [10,32,47,52,63].

1602.3. Preparation of TPx substrates

161The concentration of a commercial H2O2 standard solution was162determined based on its absorbance (e240 = 43.6 M�1 cm�1 [31])163and stock solutions freshly prepared in buffer for each experiment.164DHLA stock solutions were prepared in DMSO and stored at �80 �C165to prevent oxidation [29]. DHLA concentration was freshly deter-166mined for each experiment byDTNB assay (e412 = 14.15 mM�1 cm�1

167[18]). GSH stock solutions were freshly prepared in buffer for each168experiment.Fig. 1. Structure of TPx mimics.

Fig. 2. Catalytic cycle of TPx mimics. For TPx activity, compounds redox cycle tocatalyse the reaction of peroxides with thiol species. For this cycle, each TPx mimic(M = Se, Te) initially undergoes oxidation by H2O2. The oxidised mimic is thenreduced by a cellular thiol (R-SH) to complete the catalytic cycle and regenerate theTPx mimic in its reduced state. Overall the TPx mimic is unchanged, while the rateof reaction (H2O2 + 2R-SH? 2H2O + R-SS-R) is increased.

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169 2.4. Cell culture

170 A549 cells were cultured in a Healforce incubator under a171 humidified atmosphere containing 5% CO2/95% air at 37 �C. DMEM,172 supplemented with 2% v/v FBS and 1% v/v antibiotic–antimycotic173 solution, was used for maintenance culture.

174 2.5. Determination of relative hydrophobicity

175 TPx mimics were initially dissolved and diluted in acetonitrile176 (100% v/v) to a concentration of 100 lM. An aliquot (20 lL) of each177 solution was then injected into the HPLC system under isocratic178 conditions (75:25 v/v acetonitrile:water) over 15 min with a flow179 rate of 2.5 mL/min; compound elution was detected at 270 nm.

180 2.6. Electrochemical determination of TPx activity

181 The H2O2 electrode was initially calibrated using a range of182 H2O2 concentrations (0–1.5 mM) in a stirred buffer (50 mM KPi,183 1 mM EDTA, pH 7.0) at 37 �C. Over this range a linear relationship184 was observed between current and H2O2 concentration185 (R2 > 0.999). TPx activity was then evaluated as previously186 described [26] with minor modifications. Briefly, the TPx mimic187 (dissolved in DMSO) was added to an incubation buffer (1 mM188 GSH or DHLA, 50 mM KPi, 1 mM EDTA, pH 7.0) at 37 �C. The stock189 concentrations of TPx mimics were adjusted to maintain the DMSO190 concentration in the assay at 0.5% v/v. Once a stable baseline was191 achieved, H2O2 (1 mM) was added and the electrode response192 recorded for 5 min. The electrode response was then transformed193 into H2O2 concentration by interpolation from the calibration194 curve. The rate of H2O2 consumption was plotted against TPx195 mimic concentration and the gradient of the line of best fit deter-196 mined the pesudo zero order rate constant kobs. As the background197 rate (y axis intercept) was well defined by multiple measurements,198 the mean value of the intercept (15.1 ± 0.9 lM/min for GSH and199 16.7 ± 1.3 lM/min for DHLA) was fixed for linear regression.

200 2.7. MTT cytotoxicity assays

201 A549 cells were seeded at a density of 10,000 cells per well into202 96 well plates. After overnight incubation the media were replaced203 with either fresh DMEM or DMEM containing TPx mimics. The TPx204 mimics were initially dissolved in DMSO, and the solution then205 diluted into DMEM to give a final DMSO concentration of 0.1% v/206 v, which was kept constant for all treatments and controls. Follow-207 ing 24 h incubation, cell number was assessed via MTT assay208 (0.4 mg/mL MTT in DMEM, [43]). For cytotoxicity determination209 cell viability was expressed as a percentage of untreated controls.

210 2.8. Antioxidant versus prooxidant activity

211 Cell seeding and cell viability measurements were performed as212 described in Section 2.7. Treatments consisted of fresh DMEM,213 DMEM containing H2O2, or DMEM containing H2O2 and a non-214 cytotoxic concentration of TPx mimic (1 lM) for 4 h. All solutions215 contained a fixed DMSO concentration of 0.1% v/v. An antioxidant216 effect was defined as a shift in the cell survival concentration–re-217 sponse curve to the right, indicating that a higher concentration218 of H2O2 was required to induce cell death. A prooxidant effect219 was defined as a leftward shift in the concentration–response220 curve, indicating the compounds were augmenting H2O2 toxicity.

2212.9. Hoechst 33342 and propidium iodide double labelling cytotoxicity222assays

223A549 cells were seeded into 96 well plates at a density of 5000224cells per well and left to adhere for 24 h prior to treatment. TPx225mimic and H2O2 treatments were as described in Section 2.8. Post226treatment, the media were aspirated and replaced with Hoechst22733342 (2 lg/mL in DMEM) and incubated for 10 min. Propidium228iodide (50 lg/mL in DMEM) was then added for a further 5 min.229The cell monolayer was then washed with PBS (containing calcium230and magnesium) and media added for live cell imaging. Images231were acquired using a �20 objective with acquisition times of232500 ms for propidium iodide and 400–800 ms for Hoechst 33342.

2332.10. Data analysis

234All data were analysed and graphed using Origin Pro 2015 soft-235ware (OriginLab Corporation, Northampton, MA). Linear regression236and error were calculated using the Levenberg–Marquardt algo-237rithm with direct weighting [71] typically over 4 TPx mimic con-238centrations (n = 3 per concentration); for IC50 determination239curves were fitted using an orthogonal distance regression algo-240rithm [71], values were expressed as the mean ± SEM of individu-241ally fitted curves (n = 6). Principal component analysis and242graphing was performed using the Matlab software platform243(The MathWorks Inc., Natick, MA).

2443. Results

2453.1. TPx mimic relative hydrophobicity

246HPLC retention time (k0) was measured in order to quantify the247relative hydrophobicity of the TPx mimics. Under isocratic flow248conditions all of the compounds eluted between 3 and 11 min249(Fig. 3, Table 1). The sequential addition of methylene groups250resulted in a linear increase in logk0 across both the S and T series251(regression coefficient r2 > 0.999 for both series). In comparison to252the Se compounds, Te analogues were slightly more hydrophobic,253with Te atom substitution having an approximately equivalent254contribution as the addition of a methylene unit. As a result of255the similar hydrophobic contributions to logk0 from both Te substi-256tution and methylene groups, the pool of TPx mimics therefore257possessed pairs of compounds that were matched in either chain258length (e.g. S4:T4) or hydrophobicity (e.g. S5:T4).

2593.2. TPx mimic activity

260Using GSH as a substrate, only S7 of the S series exhibited activ-261ity (Fig. 4, Table 1). None of the others displayed measurable cat-262alytic activity within the detection limit of the assay (kobs of263approximately 0.1 min�1 with 4 concentrations per compound,264n = 3). The T series was also mostly inactive, with only T4 possess-265ing GPx activity. As only one member of each series displayed GPx266activity, there was no evident relationship between chain length267and catalysis. However it was notable that for the S series GPx268activity was only evident for the compound with the longest alkyl269chain (S7), while for the T series it was displayed by the compound270with the shortest alkyl chain (T4). This could indicate that the SAR271which determines GPx activity for these analogues was different272for Se compared to Te based compounds.273Switching from GSH to DHLA as the thiol substrate, enhanced274activity and trends in SAR became apparent. While S4 still exhib-275ited no significant activity, the other members of the S series276showed increasing activity with chain length, with a 2-fold277increase in kobs between S5 and S7. For the T series the reverse

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278 SAR was observed; T4 and T5 exhibited similar DHLA peroxidase279 activity, and activity then decreased with chain length, resulting280 in an overall 3-fold decrease in kobs between T5 and T7. Cross com-281 parison of the GSH and DHLA data revealed that S7 was equally282 active using either GSH or DHLA, whereas T4 was 3-fold more283 active using DHLA. Therefore clearly identifiable SAR trends were284 exhibited for DHLA, but there was no relationship between GSH285 kobs and DHLA kobs.

286 3.3. Cytotoxicity SAR

287 In cytotoxicity studies using the A549 cell line the S series288 displayed low cytotoxicity, with IC50 values ranging between 110289 and 220 lM (Table 1). The T series was moderately cytotoxic,

290displaying IC50 values in the range 10–30 lM. To investigate rela-291tionships between cytotoxicity, hydrophobicity and TPx activity,292the IC50 values for the combined dataset were plotted against k0

293and kobs for GSH and DHLA (Fig. 5).294For k0 trends were observed within each series; for the S series295IC50 increased as the compounds became more hydrophobic, while296for the T series IC50 decreased with increasing hydrophobicity.297However there was no relationship for the combined S and T data-298set, as compounds with approximately equal hydrophobicity (e.g.299S5 and T4) displayed disparate cytotoxicities, demonstrating that300hydrophobicity was not a predictor of cytotoxicity.301There was no relationship between IC50 and GSH kobs. T5–T7302exhibited moderate cytotoxicity but displayed no GPx activity,303while T4 possessed the highest GPx activity of all the compounds,

Fig. 3. The effect of chain length on TPx mimic hydrophobicity. The extent of drug uptake and partitioning within cells can potentially be modulated by each compounds’hydrophobic character. Therefore relative hydrophobicity between the S series (A) and T series (B) was established by measuring compound retention time (k0) via isocraticHPLC. The SAR demonstrated a linear trend between chain length and logk0 for both series (C).

Table 1TPx mimic SAR data. For k0 values are mean ± SEM (n = 3); for kobs error is the calculated error in the line of best fit (typically four concentrations with n = 3 per concentration); forIC50 determination values are mean ± SEM of individually fitted IC50 curves (n = 6).

TPx mimic k0 (min) GSH kobs (min�1) DHLA kobs (min�1) IC50 (lM) H2O2 IC50 (lM)

S4 3.21 ± 0.01 0.033 ± 0.015 0.023 ± 0.013 220 ± 30 1148 ± 43S5 4.22 ± 0.01 0.017 ± 0.019 0.140 ± 0.011 190 ± 20 963 ± 75S6 5.72 ± 0.01 0.006 ± 0.004 0.139 ± 0.021 130 ± 10 950 ± 48S7 7.84 ± 0.01 0.306 ± 0.029 0.294 ± 0.032 110 ± 8 1056 ± 100T4 3.97 ± 0.01 0.415 ± 0.036 1.141 ± 0.072 10 ± 3 416 ± 52T5 5.43 ± 0.03 �0.003 ± 0.036 1.235 ± 0.041 16 ± 4 300 ± 17T6 7.41 ± 0.01 �0.038 ± 0.054 0.779 ± 0.052 19 ± 3 1197 ± 123T7 10.32 ± 0.06 �0.133 ± 0.134 0.444 ± 0.046 30 ± 3 1143 ± 169



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304 but had similar cytotoxicity to the other members of the T series.305 For the S series only S7 exhibited GPx activity, but all displayed306 low cytotoxicity. Therefore GSH kobs was unrelated to IC50.

307A strong correlation was observed between DHLA kobs and IC50,308with increases in kobs resulting in progressive increases in cytotox-309icity. The relationship was not linear but described a curve

Fig. 4. SAR between TPx activity and chain length. TPx activity (kobs) for the S and T series was established via the electrochemical determination of the rate of H2O2 reduction[26]. Data show the rate of H2O2 reduction over a range of drug concentrations for the S series with GSH (A) and DHLA (B) and the T series with GSH (C) and DHLA (D). Valuesfor kobs using either GSH (E) or DHLA (F) as the thiol substrate were then calculated from the gradient of the line of best fit in (A–D). Data represent mean ± SEM (n = 3). ⁄kobs issignificantly different from zero (p < 0.05).



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310 between asymptotes at 290 ± 65 and 10 ± 5 lM and was therefore311 fitted to an exponential function (IC50 = 10 + 280e�5(DHLA kobs),312 r2 = 0.987). The fit indicated DHLA kobs was a good predictor of313 IC50, with compounds with low DHLA peroxidase activity (DHLA314 kobs < 0.4 min�1) displaying low cytotoxicity (>100 lM), and cyto-315 toxicity then increasing rapidly with DHLA kobs until a plateau in316 IC50 at approximately 10 lM.

317 3.4. Antioxidant versus prooxidant SAR

318 For both the S and T series no rightward shift in the H2O2 con-319 centration–response curves were observed, indicating that none of320 the compounds were acting as functional antioxidants against321 H2O2 induced stress (Fig. 6). No leftward shift in the concentra-322 tion–response curves was observed for the S series, revealing that323 these compounds also lacked functional prooxidant activity. For324 the T series T6 and T7 showed no significant change in IC50, while325 T4 and T5 significantly shifted the IC50 for H2O2 to the left, demon-326 strating that both T4 and T5 were functioning as prooxidants.327 To investigate if this prooxidant action could be attributed to328 either hydrophobicity or TPx activity, k0, GSH kobs and DHLA kobs329 were plotted against the ratio of the change in H2O2 IC50 (Fig. 6).330 There was no relationship between hydrophobicity and IC50 ratio331 (Fig. 6), with the active compounds T4 and T5 possessing interme-332 diate k0 values. Similarly no correlation was observed between GSH333 kobs and IC50. S7 and T4 possessed the only significant GPx activity,334 but T4 acted as a prooxidant while S7 was inactive. In addition T5335 displayed higher prooxidant activity than either S7 or T4, but pos-336 sessed no measurable GPx activity.337 A relationship could be identified between DHLA kobs and IC50.338 Compounds with DHLA kobs > 1 min�1 (T4 and T5) augmented339 H2O2 toxicity, while compounds with DHLA kobs values below this340 threshold were inactive. Although there were insufficient data to341 fully define the function, this relationship could be fitted to the ini-342 tial stages of a concentration–response curve. Hence the curve fit343 provided an indication that it may be possible to use DHLA kobs344 as a predictor of prooxidant activity with H2O2.

345 3.5. Multivariate analysis

346 To further identify interactions between the experimental vari-347 ables we conducted a principal component analysis of the dataset348 (Fig. 7). The majority of the variance in the dataset (>84%) could be349 explained using 2 principal components (PC1 and PC2). A scatter-350 plot of PC1 versus PC2 revealed that the S and T series could be351 grouped into 3 discreet clusters. All the S series possessed negative352 scores in both PC1 and PC2. T4 and T5 were separated from the S353 series along the PC1 axis, with positive rankings in PC1 but similar354 PC2 values, whereas T6 and T7 were separated along the PC2 axis.

355Examining the contributions of the experimental factors to the356principal components, the basis for the observed clustering357became apparent. PC1, which accounted for 51% of the total vari-358ance, was predominantly defined by contributions from DHLA kobs,359then H2O2 IC50 and IC50. Hence T4 and T5, which possessed the360highest DHLA kobs values and displayed prooxidant activity, were361clearly separated from the remaining members of the dataset in362this principal component. PC2, which accounted for 33% of the363total variance, was defined by contributions from k0, then GSH kobs364and IC50. Here the substantial hydrophobic character of T6 and T7,365coupled to their low IC50 values, resulted in a strong positive rank-366ing in PC2 which separated them from the rest of the dataset.

3674. Discussion

3684.1. SARs for GSH kobs and DHLA kobs

369When comparing kobs for the S and T series, with GSH as the370substrate only S7 and T4 displayed measurable rates, therefore371there was no observable trend in activity. In contrast, with DHLA372as the substrate, TPx activity increased with chain length for the373S series and decreased with chain length for the T series. A possible374explanation for these divergent SARs lies in the effect of the alkyl375substituent on the compounds’ catalytic mechanism. For the rela-376tively inactive Se compounds the electron donating effect of the377alkyl group may act to increase the nucleophilicity of the Se atom,378thus enhancing the overall rate. While such through bond induc-379tive effects are normally considered short range, electron donation380can also be transmitted through space [23] and increases in alkyl381chain length have been reported to have long range effects in other382catalytic mechanisms [62]. For the T series the reaction with DHLA383was faster and alkyl chain elongation had a negative impact upon384kobs. For these compounds it is likely that the steric shielding of the385alkyl group predominated over induction effects, with increasing386chain length progressively blocking access to the Te centre and387so reducing kobs.388Examining the effect of changing thiol substrate, S5–S6 and T4–389T7 preferentially oxidised DHLA over GSH, whereas S7 oxidised390both thiols at approximately the same rate. Therefore, for most391of the mimics, the choice of thiol substrate had a major impact392upon the overall catalytic rate. This contributes towards an under-393standing of the mimics’ pharmacological activity, as the extent to394which they act as antioxidants or prooxidants within the cell395depends upon which thiol species is oxidised. While the present396study only examined two thiols, the results indicate that it may397be possible to control thiol recognition by structural alterations398to the groups attached to the Se or Te centre. This has the potential399to afford compounds with a range of pharmacological properties400depending upon their specificity for different biological targets.

Fig. 5. Cytotoxicity correlates with DHLA kobs. To establish if there was a relationship between cytotoxicity and either k0 , GSH kobs or DHLA kobs, IC50 values were derived byincubating A549 cells with TPx mimics (0–100 lM) for 24 h. To establish SARs, the IC50 values were then plotted against (A) k0 , (B) GSH kobs, (C) DHLA kobs. Data representmean ± SEM (n = 6 for IC50 and n = 3 for k0 and kobs).



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401 4.2. DHLA peroxidase activity correlates with cytotoxicity

402 While much attention has been placed on the GPx activity of403 organoselenium compounds, there has been less focus on deter-404 mining their cytotoxic mechanisms. In general organoselenium405 compounds are considerably less toxic than their organotellurium406 counterparts [16,19,55,58], but the mechanism for TPx mimic cyto-407 toxicity is unknown [48,56]. Our data indicate that the cytotoxicity408 of the S and T series does not correlate with hydrophobicity.409 Therefore it is likely that the main factor determining TPx cytotox-410 icity is an interaction with one or more specific cellular targets,

411rather than the extent to which the compounds are taken up into412cells. As there was a correlation with DHLA kobs, but not with GSH413kobs, one possibility is that the compounds utilise endogenous ROS414to directly oxidise cellular DHLA and induce toxicity. Alternatively415DHLA kobs may be a predictor of cytotoxicity, with the TPx mimics416acting on a separate target which exhibits the same SAR trend.417For either scenario, the equation IC50 = 10 + 280e�5(DHLA kobs) allows418the prediction of cytotoxicity for the present structural analogues.419The extent to which this equation will predict cytotoxicity against420other cells, or if it can be applied to TPx mimics with different421organic substituents, has yet to be determined. If the relationship

Fig. 6. T4 and T5 act as prooxidants in combination with H2O2 induced stress. To determine if the TPx mimics could act as antioxidants or prooxidants, A549 cells wereincubated with either H2O2 alone (control) or H2O2 in combination with TPx mimics (1 lM) for 4 h. An antioxidant effect was defined as a shift in the H2O2 IC50 curve to theright, a prooxidant effect as a shift in the H2O2 IC50 curve to the left. IC50 plots represent mean (n = 6) nonlinear regression curves for H2O2 with the S series (A), and with the Tseries (B). To evaluate the antioxidant or prooxidant effect, the ratio of the change in IC50 from the H2O2 control (A and B) was plotted against (C) k0 , (D) GSH kobs, (E) DHLA kobs.To confirm cell death, in a separate experiment cells were double labelled with Hoechst 33342 and propidium iodide and cell death quantified by fluorescence microscopy.Fluorescence micrographs show a composite image of brightfield (grey), Hoechst 33342 (blue) and propidium iodide (red) for: control cells (F), T4 (1 lM, G), H2O2 (1 mM, H),and H2O2 (1 mM) with T4 (1 lM, I) for 4 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



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422 does extend to other TPx mimics, then DHLA kobs has the potential423 to provide a convenient approach for predicting the cytotoxicity of424 new organoselenium and organotellurium based compounds.

425 4.3. Antioxidant versus prooxidant activity

426 To establish if the compounds act as either antioxidants or427 prooxidants, cells were challenged with H2O2 and the effect of428 TPx mimics on the peroxide concentration–response curve moni-429 tored. At a non-toxic concentration (1 lM) none of the compounds430 acted as antioxidants. However T4 and T5 acted as potent prooxi-431 dants, increasing the cytotoxic effects of H2O2 administration.432 Prooxidant activity did not correlate with hydrophobicity, provid-433 ing no evidence that drug uptake and intracellular distribution434 contributed towards prooxidant action. Similarly GPx activity did435 not correlate with prooxidant activity. A potential relationship436 was identified for DHLA peroxidase activity, with the two prooxi-437 dants T4 and T5 also possessing the highest DHLA kobs values. As438 for the cytotoxicity SAR, it is feasible that DHLA oxidation was439 the mechanism of prooxidant action, but at present there are insuf-440 ficient data to identify DHLA as a cellular target.

441 4.4. DHLA as a target for TPx action

442 The correlation between DHLA kobs and TPx activity suggests the443 possibility that TPx mimics directly oxidise DHLA within cells, and

444that this reaction determines both cytotoxicity and prooxidant445action. Caveats are that there is no evidence confirming DHLA as446a cellular substrate and that mechanistic links between DHLA oxi-447dation and cell death are not well characterised. However there are448several pathways by which DHLA oxidation could contribute449towards cellular dysfunction. DHLA is a stronger antioxidant than450its oxidised form, lipoic acid (LA) [3,42]. Therefore the conversion451of DHLA to LA would reduce the cell’s overall antioxidant capacity,452a factor which may contribute towards prooxidant action. DHLA is453also an essential co-factor for enzymes involved in energy metabo-454lism [49] and LA is rapidly reduced back to DHLA at the expense of455NADH and NADPH [3]. Hence loss of NADH, NADPH and ATP could456contribute towards cytotoxicity [5,7,51,53]. DHLA also exerts a457protective function by binding ‘free’ (loosely chelated) metal ions,458blocking their accessible coordination sites and thereby inhibiting459metal catalysed cellular damage [6]. As DHLA is a more effective460chelator of labile metals than LA [37,46,61], disruptions to metal461ion homoeostasis and the release of Fe and Cu into the cytosolic462pool could explain both the cytotoxic and prooxidant effects of463TPx mimic administration.

4644.5. Comparison with known TPx mimics

465The majority of prior studies with TPx mimics have focused on466their antioxidant effects against lipid peroxides and less is known467about their activity against H2O2 stress [22,24,25,65]. Where

Fig. 7. Multivariate analysis of TPx mimic action. A principal component analysis of the dataset was conducted to identify relationships between the experimental variables.The majority of the variance could be explained by the first 2 principal components (A). Scatterplot analysis of PC1 versus PC2 indicated that the data clustered into 3 discreetgroups (B). The contribution of each experimental variable to PC1 (C) and PC2 (D) is indicated by the loading score.



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468 H2O2 has been studied, some TPx mimics exhibit prooxidant action469 [25] while other compounds have demonstrated an antioxidant470 effect [65]. None of the TPx mimics studied here displayed antiox-471 idant properties towards H2O2, possibly due to their low GSH kobs472 values. For comparison, diphenyl ditelluride has previously shown473 protection against H2O2 administration [65] and it also possesses a474 3-fold higher GSH kobs (1.55 ± 0.18 min�1 [26]) than any of the475 compounds in the S and T series. Therefore GSH kobs may prove a476 useful measure of antioxidant outcomes.

477 4.6. Future perspectives – implications for drug design

478 The present data provide insight into the parameters necessary479 to optimise TPx mimic activity. While the majority of previous480 studies have focused on examining interactions with a specific481 thiol, typically GSH or a GSH surrogate, there is obviously value482 in exploring activity with alternative thiol substrates. Examining483 only 2 potential thiol targets, the multivariate analysis identified484 3 discreet clusters which could form the basis for future investiga-485 tions. The S series displayed little pharmacological activity within486 the parameters used in this study, while the grouping of T4–T5487 and T6–T7 along PC1 and PC2 respectively indicated that it should488 be possible to explore this principal component space to design489 therapeutics that display cytotoxicity but no prooxidant activity490 (T6–T7), or a combination of cytotoxicity and prooxidant action491 (T4–T5). While none of the compounds displayed antioxidant func-492 tion, in the future a greater understanding of TPx mimic SARs may493 allow thiol substrate specificity to be controlled, which has the494 potential to allow the selection of the overall antioxidant versus495 prooxidant outcome of drug administration. For antioxidant appli-496 cations it may prove necessary to maximise the ratio between GSH497 kobs, a measure of antioxidant activity, and DHLA kobs, a predictor of498 prooxidant action. Conversely the reverse may prove necessary for499 compounds designed to have a prooxidant role. In addition to GSH500 and DHLA, multiple cysteine containing proteins have been impli-501 cated in redox signalling [64], and the H2O2 electrode assay could502 also facilitate the design of redox therapeutics which recognise503 specific thiol targets within the cell. Looking towards the future,504 this approach may lead to the realisation of new drugs specifically505 tailored for the treatment of a range of diseases.

506 Conflict of interest

507 None.

508 Acknowledgement

509 BZ was supported by a scholarship from the School of Phar-510 macy, University of Otago.

511 References

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